Netrin1 is required for post-crossing longitudinal axon guidance in the hindbrain. A , B , To trace post-crossing axonal trajectories, hindbrain open-book preparations of E12.5 control (Netrin1^+/+, Netrin1^+/−) and Netrin1^−/− mutant embryos were labeled with diI at r2. DiI crystals were placed at a mid-lateral position (data not shown) to label several subpopulations of commissural axons. In control embryos, ventral bundles (VBs) of post-crossing axons turned anteriorly to grow almost parallel to the midline ( A ). A bundle that turned at a dorsal position (*) and other axons that continued dorsally were also observed, but not further analyzed. In Netrin1^−/− mutant embryos, many post-crossing axons turned anteriorly but projected at greater angles away from the midline ( B ). A′ , B′ , Schematic diagrams of commissural axon trajectories, as visualized by diI labeling, showing the deviation of post-crossing axons at angles away from the midline in Netrin1^−/− mutant embryos ( B′ ) compared with controls ( A′ ). C , Summary graph shows that the end angles of post-crossing axons (yellow arrows) were significantly wider in the ventral bundle of Netrin1^−/− mutant embryos compared with their control littermates (normalized angle: Net^+/+/Net^+/−, −10.17 ± 1.95, n = 154 axons of 10 embryos; Net^−/−, −32.15 ± 1.97, n = 126 axons of 9 embryos; ****p < 0.0001). To normalize the angles, they were measured relative to the midline. Quantification of the end angle did not show any significant differences between the wild-type and Netrin1 heterozygous embryos (normalized angle: Net^+/+, −8.87 ± 1.76, n = 63 axons of 5 embryos; Net^+/−, −11.07 ± 3.08, n = 91 axons of 5 embryos; p = 0.6). D , E , Spinal cord commissural axons were labeled with diI crystals in the forelimb level. These axons turned anteriorly immediately after crossing the midline and made longitudinal trajectories parallel to the midline. Quantification of the end angle of these axons did not show any significant difference between the control embryos and Netrin1^−/− mutants (normalized angle: Net^+/+/Net^+/−, −0.375 ± 0.2489, n = 40 axons of 5 embryos; Net^−/−, −1.08 ± 0.684, n = 30 axons of 5 embryos; p = 0.3; F ). The end angles of post-crossing axons were not significantly different between Netrin1^+/+ and Netrin1^+/− embryos, therefore Netrin1^+/+ and Netrin1^+/− embryos together were considered as the control group. A, Anterior; P, posterior; *, axons at the dorsal position. Scale bars, 100 μm.